This proposal focuses on formation of DNA-protein complexes that trigger the onset of chromosome duplication, with the long term objective of understanding molecular mechanisms that control bacterial cell growth. In bacteria, the rate of initiation of chromosome replication determines the frequency of cell duplication and requires assembly of a pre-replication complex (pre-RC), comprising multiple copies of the conserved initiator DnaA bound to an origin of replication, oriC. Our hypothesis is that the oriC sequence contains all the information required to ensure that the pre-RC is assembled at the correct time in the cell cycle at all growth rates. We base this hypothesis on our observations that: 1) E. coli oriC contains high affinity DnaA binding sites that are occupied throughout the cell cycle, forming a bacterial Origin Recognition Complex (ORC); 2) The ORC sites flank arrays of lower affinity sites which interact with DnaA to form the pre-RC only at the time of initiation of chromosome replication; thus occupancy of low affinity sites determines initiation timing; 3) Low affinity sites are targets for multiple mechanisms that regulate initiation by modulating DnaA binding; and 4) DnaA cannot occupy low affinity sites in the absence of ORC. Based on these observations our goals for this proposal are to define how arrangement of high and low affinity DnaA binding sites in E. coli oriC directs ORC to pre-RC transition and regulates timing of initiation during the cell cycle, and to understand how this transition may be regulated by growth rate-dependent mechanisms. The Specific Aims are to: 1) Test the hypothesis that loading and stable occupation of low affinity DnaA recognition sites is dependent on proximal ORC sites; 2) Test the hypothesis that loading of phased arrays of low affinity DnaA recognition sites is a late stage of pre-RC formation that determines the requirement for DnaA-ATP and regulates initiation timing during the cell cycle; and 3) Test the hypothesis that growth rate regulated binding of Fis and IHF to oriC modulates the level of DnaA required for low affinity site occupation. Accomplishing these aims will advance our understanding of how sequence information in bacterial origins directs precisely timed, cell cycle specific complex assembly. This information will improve our understanding of growth regulation in both enteric and non-enteric pathogens. Studies described here will also aid in the identification of new targets appropriate to guide the design of novel cell growth inhibitors for bacterial pathogens.